Method and device for concentrating selected groups of microorganisms

ABSTRACT

A method and device are described to concentrate target organisms from a mixture of organisms. Beads ( 1 ) made of material such as nylon, polystyrene or glass are coated with antibodies of specific microorganisms. The beads ( 1 ) are contained in an enclosure ( 2 ) surrounded by grid material ( 4 ). The pore size of the grid is smaller than the size of the beads, to assure that the beads stay within the grid material and larger than the size of the microorganisms to allow the interaction of the microorganisms with the beads. A rod ( 5 ) is attached to the upper part of the enclosure ( 2 ) allowing the agitation of the device inside he growth medium containing the target organisms.

This application is a 371 of PCT/US99/18618 filed Aug. 23, 1999 whichclaims benefit of Provisional application Ser. No. 60/097,627 filed Aug.24, 1998.

BACKGROUND

1. Field of Invention

This application is based on provisional patent application Ser. No.60/097,627, filed Aug. 24, 1998.

The present invention relates to products and processes used for thedetection of microbes in a sample. More specifically, the presentinvention provides a method and device for aiding in the detection ofthe presence of specific microbial contamination in food samples,clinical specimens and other products.

2. Prior Art

It is necessary to test various substances, such as foods, beverages,pharmaceuticals, cosmetics, water, and body fluids for microbialcontamination, especially with certain pathogenic bacteria. Recentoutbreaks of foodborne illness, implicating a variety of foodscontaminated with pathogenic bacteria, such as E. coli 0157:H7,Salmonella, Listeria, Campylobacter jejuni, and Cyclospora, haveunderscored the need for rapid methods for microbiological analysis.Microbiological analysis is critical for assessment of safety andquality, to determine efficiency of manufacturing, and conformance withregulations.

The increased scope, significance, and need for microbiological testingserved to reveal the limitations and drawbacks of conventional methods.Classical methods for determining the presence of pathogenic bacteria insamples are taking typically several days to perform. It is desired toprovide rapid detection of especially pathogenic bacteria causingillnesses.

Since the desired sensitivity for most assays for pathogenic bacteria isless than one such organism in 25 grams of product, most testing methodsrely on an initial enrichment step. The indigenous microflora that isusually present in many foods at high levels often interferes with theselective isolation and identification of pathogenic bacteria. Foodprocessing such as heating, cooling, drying, freezing, addition ofpreservatives and other causes can sub-lethally injure bacterial cells.These injured cells are extremely sensitive to the ingredients used inselective microbiological media. Therefore, in many assays the processstarts with pre-enrichment, in which the sample is incubated in anutritious, non-selective medium to allow the resuscitation of injuredor stressed bacteria. This step is followed by a selective enrichmentstep where the bacteria of interest are allowed to grow while theindigenous microflora is suppressed. The enrichment procedure isfollowed either by conventional plating methodology or a variety of moremodern and rapid methods such as DNA amplification or immunoassay.

It is therefore desired to separate at an early stage the targetorganisms from the other flora present in the product. One such approachis the utilization of the immuno-magnetic separation technique,involving the utilization of immuno-magnetic particles specific for thetarget organisms. Magnetic beads with antibodies affixed to theirsurfaces are mixed with the sample containing the target organism. Thisorganism will bind to the bead surfaces via the antibodies. Theorganism-bead complex is pulled out of the solution by a magnet, toconcentrate the microorganisms.

U.S. Pat. No. 4,230,685 describes magnetically responsive microsphereshaving protein A associated with the outer surface. The microspheres arereacted with antibodies selective to cells, bacteria or viruses to beseparated from a mixed population. The microorganism will attach to theantibody and thereby to the microspheres, and the microspheres are thenused in a magnetic separation procedure. The preferred microspheres areprepared from a mixture of albumin, Protein A, and magnetic particles.The microspheres are prepared so that the Protein A is present in theexterior surface of the antibody binding. U.S. Pat. No. 4,695,393describes a process for the preparation of such magnetic beads, whichcan be used in separation of microorganisms.

U.S. Pat. Nos. 5,491,068 and 5,695,946 describe a method characterizedby antibody capture of the organism of interest by the application ofspecialized magnetic beads. It entails the incubation of the capturecells to form colonies; removal of material from the colonies withcolony lift membrane; and detection of the colony material on themembrane sheet by the use of labeled antibodies, PCR or nucleic acidprobes. The main problem with this method is the low sensitivity of oneorganism per gram. This low sensitivity is inherent in the methodologyand is 50–100 fold lower than the desired sensitivity for most foodpathogens.

U.S. Pat. No. 4,677,055 describes a process for concentrating bacteriautilizing magnetic gel to which anti-specific antigenic determinantantibodies are coupled. It involves the steps of obtaining mediumcontaining the organisms possessing specific antigenic determinants andbringing them in contact with particles of the magnetic gel. This stepis followed by the separation of the gel from the medium by magneticmeans and inoculation into new medium.

In general there are a number of problems associated with magneticbeads. One such problem results from the small size of such beads (3–10μm) and the large volume of the medium (250–3,000 ml). As a result it isimpossible to remove the magnetic beads from such a large volume.Therefore, many procedures either use a lower sample volume (therebyreducing the sensitivity of the assay) or allow some time (8–18 hours)of pre-enrichment followed by the removal of 1–5 ml of solution forconcentration of the target organisms. Another problem associated withthe magnetic beads is the fact that they get coated with fat andproteins making it difficult to be collected with a magnet. The processof separating the beads from the medium and washing the unattachedbacteria is labor intensive, and creates a contamination hazard of bothlaboratory surfaces and the beads.

OBJECTS AND ADVANTAGES

It is, therefore, an object of the invention to provide a method anddevice that can be utilized with a large volume of media, to concentratea target organism. It is another object of the invention to provide amethod that is less labor intensive, more rapid and will lend itself toautomation.

Still further objects and advantages will become apparent from aconsideration of the ensuing description and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a side view of the preferred device utilized to concentratetarget organisms.

FIG. 2 shows a side view of another design of the device utilized toconcentrate target organisms.

DESCRIPTION

FIG. 1 shows the preferred embodiment of a device for the separation ofthe target organisms from a suspension containing a mixture oforganisms. Beads 1 are made of materials such as nylon, polystyrene orglass. The beads are coated with antibodies to specific microorganismssuch as Salmonella, E. coli 0157:H7 and Listeria. A cylindricalenclosure 2 is designed to contain the beads. The enclosure isconstructed from a frame 3 supporting a grid 4 covering the frame. Thegrid's pore size is smaller than the size of the beads to assure thatthe beads stay within the enclosure 2. However, the pore size is largeenough to allow bacteria to freely pass into the enclosure. A rod 5 isattached to the upper part of the enclosure. The rod 5 allows theenclosure 2 to move in the solution and for subsequent removal of thedevice from the solution.

FIG. 2 shows a different design of the device. The beads 11 coated withantibodies are contained in the enclosure 12 made of a grid 13, shapedlike a tea bag. A non-wicking string 14 is attached to the upper part ofthe enclosure 12 allowing movement of the enclosure 12 in the solution,while disallowing the solution containing bacteria to wick up thestring. The grid's 13 pore size is smaller than the size of the beads toassure that the beads stay within the enclosure. However, the pore sizeis large enough to allow bacteria to freely pass into the enclosure.

The food sample to be tested for the presence of the target organism ismixed with the appropriate pre-enrichment broth. The pre-enrichmentbroth is incubated at an appropriate temperature. Upon the beginning ofthe incubation period, or alternatively after several hours ofincubation, the enclosure 2 is immersed into the broth containing thesample thereby exposing the beads having immobilized thereon monoclonalor polyclonal antibodies to the selected bacteria of interest. This isaccomplished by lowering the device 2 into the solution and agitating itfor at least 30 minutes and up to several hours. This step allows cellcapture by the beads, and the creation of bead-target microbial cellcomplexes. The next step involves the separation of the beads with thebound target cells from the suspension containing the food particles andother mixed flora. This is accomplished by pulling the whole device outof the solution, using the rod 5. The device is subsequently washedseveral times in sterile saline or buffer solution. The washing solutionis changed after each wash to remove non-bound organisms. Addition ofdetergents such as Tween-20 (0.51–0.1% w/v) or protamine to theincubation broth mixture usually decreases the non-specific adsorption.Tween-20 can be also used in the washing procedure to removenon-specifically bound cells. After the wash step a number of methodscan be utilized to detect the presence of the target organism. Dependingupon the particular detection method to be used, as explained below, thebeads may or may not be separated from the enclosure after the washstep.

Several detection procedures can be used in conjunction with the currentinvention to detect the presence of the microorganism of interest. Forexample, the device can be inserted into a new growth broth thatincludes a dye indicator and the changes in the dye characteristics canbe utilized to determine presence or absence of the target organism. Themicroorganisms do not need to be detached from the beads sinceattachment to the beads has no effect on their growth. Therefore cellscan continue to multiply in the appropriate medium. Alternatively thebeads can be removed from the enclosure and inoculated onto the surfaceof appropriate selective or differential agar. Another approach is toutilize an immunoassay. Most immunoassays require 10³–10⁵ cells ml⁻¹,therefore the beads should contain enough cells to perform a directimmunoassay. Similarly, this method can be combined with DNAhybridization and amplification techniques such as PCR.

As can be seen from the above disclosure, the method of the invention isparticularly characterized by the use of immunological beads containedin an enclosure to select out target microorganisms from the sample. Thebeads must be capable of effectively capturing the target microorganismsfrom the test sample, while not capturing significant numbers of otherorganisms that might be present at much higher numbers. However, theantibody used need not be totally specific to the target organism sincean additional selection step is available at the end of the assay. Theantibodies must be oriented with their binding sites outward to allowcontact between the binding portion of the antibody and the targetorganism. The size of the beads must be larger than the size of themicroorganism, to remain contained in the enclosure, while allowing thetarget organism to enter the enclosure and attach to the beads. Thecontact time between the beads and the target organism must be longenough to allow strong interaction. Several hours of interaction wasfound to yield the best results, i.e. the creation of stronginteractions to produce high capture efficiency. After the completion ofthe incubation step the beads are removed from the solution, by theremoval of the enclosure in which they are contained. The enclosure andthe beads are washed several times, and the beads are transferred intothe detection system.

CONCLUSIONS, RAMIFICATIONS, AND SCOPE

Accordingly, it can be seen that the new method and device can beutilized with a large volume of media, to concentrate a target organism,without the need to utilize only a portion of the pre-enrichment brothor a small volume of enrichment broth as required for magnetic beads.The invention provides a method and device that is less labor intensive,more rapid and lends itself to automation. Many different designs, forcontaining the beads during the various steps of the assay, can beutilized.

Obviously, many modifications and variations of the present inventionare possible in light of the above techniques. Although the descriptionabove contains many specificities, these should not be construed aslimiting the scope of the invention but as merely providingillustrations of some of the presently preferred embodiments of thisinvention. The invention may be practiced otherwise than as specificallydescribed.

1. A method of separating target microorganisms from a suspension,comprising: immersing a plurality of beads coated with at least oneantibody material into the suspension, said beads being contained withinthe interior space of an enclosure having a pore size smaller than thesize of said beads and larger than the size of the microorganisms,suspending the enclosure in the suspension thereby allowing the captureof the target microorganisms by said beads; and washing said beads toremove organisms not bound to said beads, thereby separating targetmicroorganisms from the suspension.
 2. The method of claim 1 wherein atleast one detergent is applied in said washing.
 3. The method of claim 1further comprising agitation of said enclosure holding said beads in thesuspension.
 4. The method of claim 1 wherein the time period ofagitation is at least 30 minutes.
 5. The method of claim 1 wherein thetime period of agitation extends for several hours.
 6. The method ofclaim 1 including the addition of at least one detergent to thesuspension to decrease absorption of non-specifically bound cells. 7.The method of claim 1 including the subsequent step of immersing theenclosure and beads in new growth broth.
 8. The method of claim 7including the addition of an indicator material to the new growth broth.9. The method of claim 1 including the subsequent step of separating thebeads from the enclosure followed by at least one test to reveal themicroorganisms of interest.
 10. The method of claim 1 wherein saidenclosure further comprises means attached to the enclosure for movingthe enclosure in the suspension and for subsequent removal of theenclosure from the suspension.
 11. The method of claim 10 wherein saidmeans attached to the enclosure for moving the enclosure in thesuspension and for subsequent removal of the enclosure from thesuspension is attached to an upper part of the enclosure.
 12. The methodof claim 1 wherein said enclosure is made of a grid material.
 13. Themethod of claim 3 wherein said enclosure holding said beads in thesuspension is agitated with a rod or string attached to the enclosure.14. The method of claim 1 wherein said enclosure is in the shape of ateabag.
 15. The method of claim 1 wherein said enclosure is cylindrical.